The optimal mixing ratio of Brassica napus and Brassica juncea meal improve nematode Meloidogyne hapla effects

ABSTRACT

The use of rapeseed (Brassica napus L.) or leaf mustard (Brassica juncea L. Czern) meal or both as organic fertilizer not only improves the soil environment and crop productivity by supplying nutrients but also has nematicidal effects. This study aimed to establish the optimal application levels of rapeseed and leaf mustard meal for stronger nematode control in tomato. Tomato is one of the most important solanaceous crops which is severely damaged by nematodes. At first, meal (120 g of varying mixing ratios of rapeseed and leaf mustard meal) was mixed with sterilized soil (1 kg). The optimal ratio of rapeseed:leaf mustard meal for effective nematode control was 20:100 g/kg of soil. Progoitrin and gluconapin were the most abundant glucosinolates found in rapeseed meal, while sinigrin was the most abundant in leaf mustard meal. The amount of sinigrin increased if the leaf mustard meal proportion increased in the meal mixture. Although the content of sinigrin in optimal ratio mixture of rapeseed and leaf mustard meal is lower than only leaf mustard meal, it is presumed that nematocidal effects of the mixture are better than that of the single component due to the high contents of progoitrin and gluconapin. So, we propose that rapeseed and leaf mustard meal mixture at an appropriate ratio can be used as an environmentally friendly nematocide.

KEYWORDS:

• Glucosinolate
• leaf mustard meal
• nematode management
• rapeseed meal

In greenhouse practices, almost all crops including cucumber, melon, and tomato undergo continuous cultivation and are damaged by the infection of root-knot nematodes, which reduces the yield by 30–40% approximately per year.^1,2 The excessive occurrence of root-knot nematode inhibits the growth of crops as well as damage the root systems.³ For example, root-knot nematodes (Meloidogyne spp.) parasitize the roots of crops and inhibit the growth.⁴ The effective control of nematode density not only improves the growth of plant vegetable crop but also reduces the deterioration of yield and quality.⁵ However, it is difficult to apply nematocides effectively because owing to continuous cultivation, nematode habitats are deep in the soil, and nematode density recovery is accelerated.⁶ Therefore, nematocidal control needs to be applied continuously but should not be harmful to other organisms in the environment.

Hydrolyzed glucosinolates are known to suppress the occurrence of nematodes in an environmentally friendly manner. More than 100 types of glucosinolates are known to exist as secondary metabolites in Brassicaceae species.^7,8 Glucosinolates can be degraded by enzymes such as myrosinases.⁵ These are stable compounds that dissolve in water and have been reported to act on soil organisms to reduce disease incidence in greenhouses.⁹ In particular, glucotropaeolin, glucoerucin, and sinigrin showed the strongest effect on nematicidal activities due to the glucosinolate-myrosinase system.^10–12 Glucosinolates are found in plants belonging to the family Brassicaceae, such as Brassica napus L., Brassica juncea, Brassica campestris, and Brassica rapa L. .^8,13,14

Rapeseed meal, a by-product of rapeseed (B. napus) oil production is known to be effective against nematode and soil disease because of its glucosinolate content.^15,16 Recently, Park et al.⁸ proposed that using a mixture of rapeseed and leaf mustard meal as a nematocide might improve the efficiency of nematode control by compensating for the relatively low sinigrin content of rapeseed meal. The rapeseed meal contains progoitrin and gluconapin as major glycosinolate components.

In this study, we aimed to determine the nematocidal potential of rapeseed and leaf mustard meal application, their optimal ratio mix, and the glucosinolate compositions of the two meal types to elucidate the mechanisms of their different nematode inhibitory effects.

Tomato (Solanum lycopersicum L., ‘Rutgers’) seedlings were cultivated for 20 days in a pot and transplanted into new pots containing the experimental soil. To confirm the proper application level of the meals as for efficient nematode control in tomato, firstly 1kg of sterilized soil was mixed with 0, 40, 80 and 120g/kg of rapeseed (Brassica napus) meal individually. Then, these tomato plants were inoculated with 1,000 ~ 1,500 larvae of nematode (Meloidogyne hapla)/plant and incubated in a greenhouse for 8 weeks. Secondly, 1 kg of soil was mixed with varying ratio mixtures of rapeseed (Brassica napus) and leaf mustard (Brassica juncea) meal (i.e., 120 + 0 g, 20 + 100 g, 40 + 80 g, 60 + 60 g, 80 + 40 g, 100 + 20 g, and 0 + 120 g). After 1 week, 2,000–2,500 larvae of the nematode (M. hapla) were inoculated into each experimental pots.

To determine the nematode density in the experimental soils, nematodes were isolated from 500 g of the potted experimental soils and mixed with water. The muddy water was sequentially filtered using a 200 mesh sieve, followed by a 400 mesh sieve. The separated supernatant was discarded, and sugar water (specific gravity 1.18) was added to two-thirds of the mixture. The supernatant was passed through a 500 mesh sieve. Then 10 mL of distilled water containing nematodes were collected from the sieve. To determine the nematode density, the number of nematodes under 2mL of the solution were counted using a microscope (Carl Zeiss, Germany), where the nematodes were observed under 40× magnification. Finally, the nematode density in 10 mL of the solution was calculated to be 5-fold.¹⁷

Glucosinolates were extracted as described by Park et al.⁸ Each 100 mg of freeze-dried and homogenized rapeseed and leaf mustard meal was mixed with methanol in water in a 70°C to inactivate the endomyrosinase. The collected supernatant was considered the crude glucosinolate extrat. The crude glucosinolates and sinigrin (external standard) were desulfated. The eluates were filtered and analyzed immediately using high-performance liquid chromatography (HPLC). Glucosinolate mass spectrometry (MS) was performed using an HPLC system (Agilent 1200 series, Agilent Technologies). The enhanced potential of the 10 v. Analyst® software (SCIEX) program was used to assess the tandem MS (MS/MS) peaks. The peaks were identified by comparing reference standards reported in the study by Kuszierewicz te al.¹⁸ with our results.

All biological data were analyzed using Duncan’s test after one-way ANOVA. Statistical analyzes were carried out using SPSS version 20 software (IBM, Chicago, IL, USA). Statistical differences were considered statistically significant at P ≤ 0.05.

As the results of confirming the appropriate application level of the meal for efficient nematode control in tomato, the nematode density was 153/10mL extract solution for the 80 g/kg rapeseed meal treatment, and this increased 3.6-fold and decrease 2.7-fold in soil containing 40 and 120 g/kg rapeseed meal, respectively (Table 1). Furthermore, rapeseed meal concentrations higher than 120 g/kg inhibited tomato plant growth (data not shown). Lee et al.¹⁹ reported an effect of nematode control on tomato plants treated with 100 g/kg.

Table 1. Effect of nematode control on rapeseed (Brassica napus) meal in soil experiments with tomato host plants. (Unit: No./10 mL).

Treatment (g/kg soil)^a		Number of nematodes^b
Rapeseed meal	0	1160 ± 173.5 a
	40	504 ± 48.1 b
	80	153 ± 19.2 c
	120	55 ± 6.6 d

^a1,000–1,500 nematode (Meloidogyne hapla) larvae were inoculated by drilling 2–3 cm deep holes around the roots under the different amount of rapeseed meal conditions.

^bAll data are means ± standard deviation of nine replicates. Different lowercase letters indicate significant difference at P < 0.05 using Duncan’s test.

To investigate the nematode controlling effect of the mixture of rapeseed and leaf mustard meal, the host tomato plant was transplanted into soil containing rapeseed and leaf mustard meal mixed at various ratios based on 120 g/kg. The result showed that the nematode density was approximately 664/10 mL extract solution in soil treated with 120 g/kg rapeseed meal (Table 2), whereas that of the soil treated with 120 g/kg leaf mustard meal was 3.4 times lower (Table 2). Besides this, the nematode density was remarkably lower in soils treated with specific ratio mixtures of rapeseed and leaf mustard meal than in those treated with rapeseed meal or leaf mustard meal alone. The nematode density decreased as the ratio of leaf mustard meal increased in the mixture (Table 2). Specifically, mixtures containing an appropriate ratio of both types of the meal (20 g rapeseed meal plus 100 g leaf mustard meal per 1 kg of soil) showed a synergistic effect that was approximately 13.6 and 4 times greater than that of rapeseed alone and leaf mustard meal alone, respectively. However, the effect of the inhibition of nematode densities differed according to the mixed ratios of rapeseed meal and leaf mustard meal. This result suggested that even mixed preparations having a high ratio of rapeseed meal to leaf mustard meal can be used as an effective nematicide control agent.

Table 2. Nematode controlling effects of mixtures of rapeseed (Brassica napus) and leaf mustard (Brassica juncea) meal in soil experiments with tomato host plants. (Unit: No./10 mL).

Treatment (g/kg soil)^a		Number of nematodes^b
Negative control	no treatment	2320.4 ± 347.0
Rapeseed meal	120	664.4 ± 28.3 a
Leaf mustard meal	120	192.0 ± 12.6 b
Rapeseed meal + Leaf mustard meal	100 + 20	94.7 ± 7.4 c
	80 + 40	87 ± 5.3 c
	60 + 60	74.9 ± 5.5 d
	40 + 80	56.0 ± 3.8 e
	20 + 100	48.8 ± 4.4 e

^a2,000–2,500 nematode (Meloidogyne hapla) larvae were inoculated by drilling 2–3 cm deep holes around roots of host plants under the various ratio of rapeseed meal and leaf mustard meal conditions.

^bAll data are means ± standard deviation of nine replicates. Different lowercase letters indicate significant difference at P < 0.05 using Duncan’s test

To find the cause of the difference in nematode inhibitory effects between treatments with varying mixing ratios of rapeseed and leaf mustard meal, the glucosinolate content and components of the mixed oil of rapeseed meal and leaf mustard meal were analyzed. Total glucosinolates content of rapeseed meal alone was lowest, 32.86 μmol/g, whereas those of leaf mustard meal alone was highest, 112.77 μmol/g. In addition, it was confirmed that the content of glucosinolate increased as the amount of leaf mustard meal increased in the treatments (Table 3). Furthermore, rapeseed meal and leaf mustard meal showed different types of glucosinolate mainly. The glucosinolates of rapeseed meal consisted mainly of progoitrin (14.41 μmol/g) and gluconapin (10.26 μmol/g), and these of leaf mustard meal consisted mostly of progoitrin (104.34 μmol/g). For this reason, the content of progoitrin and gluconapin decreased as the leaf mustard meal proportion increased in the meal mixture. Moreover, the sinigrin content increased with increasing amounts of leaf mustard meal in the mixture of both types of meal (Table 3). In addition, the amounts of progoitrin, gluconapin, and sinigrin in the mixture of rapeseed and leaf mustard meal (40 + 80 g or 20 + 100 g, the optimal ratios with superior root-knot nematode density inhibitory effect) were 3 to 8 μmol/g progoitrin and gluconapin, and approximately 80 to 95 μmol/g sinigrin (Table 3).

Table 3. Glucosinolate content in rapeseed (Brassica napus), leaf mustard (Brassica juncea), or mixtures of both types of meal.

Glucosinolates	Rapeseed meal plus leaf mustard meal (g/kg soil)
	120 + 0	0 + 120	100 + 20	80 + 40	60 + 60	40 + 80	20 + 100
Progoitrin	14.4 ± 0.6 b^a	0.3 ± 0.0 e	18.4 ± 0.7 a	15.4 ± 0.9 b	8.8 ± 0.4 c	7.8 ± 0.4 c	3.5 ± 0.3 d
Glucoraphanin	0.7 ± 0.0 b	0.1 ± 0.1 d	0.9 ± 0.1 a	0.8 ± 0.0 ab	0.4 ± 0.1 c	0.4 ± 0.0 c	0.2 ± 0.0 d
Sinigrin	0.1 ± 0.0 f	104.3 ± 2.6a	26.2 ± 1.5 e	48.7 ± 1.3 d	69.7 ± 5.6 c	83.5 ± 4.4 b	95.3 ± 3.2 a
Glucoalyssin	0.5 ± 0.0 a	0.2 ± 0.0 b	0.6 ± 0.0a	0.5 ± 0.0a	0.3 ± 0.0 b	0.3 ± 0.0b	0.1 ± 0.0 c
Gluconapoleiferin	0.4 ± 0.0 b	ND^b e	0.5 ± 0.0 a	0.4 ± 0.0 b	0.2 ± 0.0 c	0.2 ± 0.0 c	0.1 ± 0.0 d
Gluconapin	10.3 ± 0.3 c	0.8 ± 0.1f	14.4 ± 0.2 a	11.8 ± 0.5b	6.8 ± 0.3 d	6.2 ± 0.3 d	3.6 ± 0.4 e
4-Hydroxyglucobrassicin	2.9 ± 0.3 b	5.1 ± 0.2 a	2.1 ± 0.5 b	1.9 ± 0.3 b	2.4 ± 0.7 b	2.8 ± 0.5 b	2.9 ± 0.5 b
Glucocochlearin	ND	ND	0.2 ± 0.0	0.2 ± 0.0	0.2 ± 0.0	ND	0.3 ± 0.0
Glucobrassicanapin	1.5 ± 0.1 b	ND	1.8 ± 0.0 a	1.51 ± 0.1 b	0.9 ± 0.0 c	0.7 ± 0.1 d	0.4 ± 0.0 e
Glucoerucin	0.2 ± 0.0 a	0.2 ± 0.0 c	0.3 ± 0.0 a	0.2 ± 0.0 ab	0.1 ± 0.1 c	0.2 ± 0.0 bc	0.2 ± 0.0 c
Glucobrassicin	0.7 ± 0.0 a	0.4 ± 0.0 cd	0.6 ± 0.0 a	0.6 ± 0.0 ab	0.4 ± 0.1 bc	0.3 ± 0.1cd	0.2 ± 0.0 d
4-Methoxyglucobrassicin	0.6 ± 0.0 a	0.5 ± 0.0 a	0.5 ± 0.0 ab	0.5 ± 0.0 ab	0.4 ± 0.1 ab	0.2 ± 0.2 bc	0.1 ± 0.0 c
Gluconasturtiin	0.6 ± 0.0 a	0.7 ± 0.0 a	0.8 ± 0.0 a	0.8 ± 0.0 a	0.7 ± 0.0 a	0.8 ± 0.1 a	0.7 ± 0.0 a
Neoglucobrassicin	ND	0.2 ± 0.0	ND	ND	ND	ND	ND
Total	32.9 ± 1.4	112.8 ± 2.9	67.1 ± 2.1	83.2 ± 2.7	91.5 ± 5.5	103.3 ± 4.4	107.6 ± 3.8

^aAll data are means ± standard deviation (μmol/g DW) of nine replicates. Bold type text indicates major glucosinolates in rapeseed or leaf mustard meal or mixtures of both. Different lowercase letters indicate a significant difference between seven treatments subjected to the same glycosinolate at P < 0.05 using Duncan’s test

^bNone determined (ND)

The cause of the difference in nematode inhibitory effect according to the mixing ratio of rapeseed and leaf mustard meal was investigated by assessing the content of glucosinolates. The glucosinolates in the mixture of rapeseed meal and leaf mustard meal (20 + 100 g), which was the most effective nematode control ratio, consisted mainly of sinigrin (95.26 μmol/g). This content of sinigrin is lower than that of rapeseed meal alone (104.34 μmol/g). Despite the lower content of sinigrin in optimal ratio mixture of rapeseed meal and leaf mustard meal, it is presumed that the effect of nematocidal control of the mixture is better than that of the alone component due to the high contents of other glucosinolates, such as progoitrin, gluconapin. The glucosinolates have been reported to have different nematicidal effects depending on the type.^12,20 A small amount of sinigrin showed a stronger nematicidal effect, while a large amount of progoitrin required. However, there was little difference in the amount of sinigrin and progoitrin required for the immobilization effect on nematode. According to,¹² progoitrin showed a low nematicidal activity but a high larval immobilization effect, and sinigrin showed high nematicidal activity. Taken together with our results, it was presumed that progoitrin in rapeseed meal maximizes the nematicidal activity of sinigrin in leaf mustard meal by immobilization on nematode.

This study showed that soil treatment using a mixture of rapeseed meal and leaf mustard meal has a remarkably superior nematocidal effect than using treatments of only rapeseed meal or leaf mustard meal. Certain mixed formulation of rapeseed meal and leaf mustard meal contain a sufficient concentration of glucosinolates as active ingredients that have excellent nematocidal effects and can be used to control the excessive occurrence of parasitic root-knot nematodes in an environmentally friendly manner. We, therefore, propose that rapeseed and leaf mustard meal, mixed at an appropriate ratio, can be used as an effective and environmentally friendly nematocide in facility house and continuous cultivation practices.

Disclosure of potential conflicts of interest

The authors are declare that they have no conflict of interest.

Additional information

Funding

This work was supported by the Cooperative Research Program for Agriculture Science and Technology Development under Rural Development Administration, Republic of Korea [Project No. PJ01212301].